A traditional Chinese medicine composition for preventing and treating drug-resistant Escherichia coli disease in chickens and its application

Abstract

The present invention relates to a Chinese medicine composition for preventing and treating drug-resistant Escherichia coli disease in chickens and its application, and belongs to the field of animal medicine technology. The Chinese medicine composition is composed of the following components in parts by mass: Gardenia jasminoides, scutellaria baicalensis, forsythia suspensa, dandelion, coptis chinensis, bupleurum, liquorice, star anise, peony bark, trichosanthes root, akebia caulis, pomegranate peel, transfer factor, wherein the transfer factor is a chicken spleen transfer factor prepared by ultrasonic wall breaking, repeated freeze-thaw treatment, centrifugation, dialysis and ultrafiltration. The present invention exerts a synergistic effect of multiple components and multiple targets through the reasonable compatibility of Chinese medicine ingredients, and shows a significant inhibitory effect on both standard Escherichia coli and mcr-1 positive drug-resistant Escherichia coli. Moreover, the composition has an extremely high cure rate and total effective rate in treating chicken Escherichia coli disease caused by mcr-1 positive drug-resistant Escherichia coli. Therefore, the composition can be used to prepare medicines and antibacterial agents for treating chicken Escherichia coli disease, and has good promotion and application value.

Description

Technical Field

[0001] The invention belongs to the technical field of animal medicine, and in particular relates to a traditional Chinese medicine composition for preventing and treating drug-resistant Escherichia coli disease in chickens and an application thereof. Background Art

[0002] Colibacillosis in chickens is a common bacterial disease caused by pathogenic Escherichia coli. It is widespread in poultry farms worldwide, especially in intensive farming environments. Chickens, especially those raised in high-density environments with poor ventilation and sanitation, are more susceptible to E. coli infection. Colibacillosis often has a high incidence rate and spreads rapidly. Once it occurs, it often leads to a series of problems such as slowed growth, decreased egg production, and increased mortality in chickens, resulting in significant economic losses to the chicken industry.

[0003] Traditionally, antibiotics have been the primary means of preventing and treating E. coli infections in chickens. Appropriate use of antibiotics can effectively inhibit the growth and spread of E. coli. However, the long-term and irregular use of antibiotics, particularly in the livestock industry, has led to an increasing problem of antibiotic resistance, resulting in the rapid development and widespread spread of drug-resistant E. coli. The emergence of drug-resistant strains has significantly reduced the therapeutic efficacy of many traditional antibiotics, or even completely rendered them ineffective, posing unprecedented challenges to clinical treatment. While research on common E. coli infections is relatively extensive, research on E. coli infections caused by drug-resistant E. coli remains relatively scarce, resulting in increasingly limited clinical treatment options and limited efficacy. Therefore, identifying effective drugs and treatments for drug-resistant E. coli infections has become a critical issue that urgently needs to be addressed. Summary of the Invention

[0004] The purpose of the present invention is to provide a traditional Chinese medicine composition for preventing and treating drug-resistant Escherichia coli disease in chickens and application of the composition in preparing a medicine for treating drug-resistant Escherichia coli disease in chickens.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a traditional Chinese medicine composition for preventing and treating Escherichia coli disease in chickens, wherein the traditional Chinese medicine composition is composed of the following components in parts by mass:

[0007] Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Transfer Factor: 0.25-0.5 parts.

[0008] Preferably, the transfer factor is chicken spleen transfer factor;

[0009] The preparation method of the chicken spleen transfer factor comprises the following steps:

[0010] (a) Wash the chicken spleen with triple-distilled water to remove impurities, then rinse with sterile saline;

[0011] (b) mincing the cleaned chicken spleen, adding 2 volumes of normal saline, and preparing a homogenate using ultrasonic cell wall disruption technology;

[0012] (c) subjecting the homogenate to repeated freeze-thaw operations, and subjecting the homogenate to high-speed homogenization and grinding each time after complete thawing;

[0013] (d) After low-temperature centrifugation, the supernatant was collected and filtered under pressure, and the obtained filtrate 1 was dialyzed to obtain filtrate 2;

[0014] (e) ultrafiltration of filtrate 2 using a 5000 molecular weight ultrafiltration membrane, and collecting the permeate;

[0015] (f) The permeate is sterilized by filtration and then freeze-dried to obtain chicken spleen transfer factor.

[0016] 3. The Chinese medicine composition according to claim 2, characterized in that the chicken colibacillosis is chicken colibacillosis caused by standard Escherichia coli or mcr-1 positive Escherichia coli.

[0017] Preferably, the chicken colibacillosis is chicken colibacillosis caused by standard Escherichia coli or mcr-1 positive Escherichia coli.

[0018] Preferably, the Chinese medicine composition consists of the following components in parts by mass:

[0019] Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

[0020] In a second aspect, the present invention provides a method for preparing a drug for preventing and treating chicken Escherichia coli disease, the method comprising the following steps:

[0021] (1) Grind 10-20 parts of Gardenia jasminoides, 10-20 parts of Scutellaria baicalensis, 15-30 parts of Forsythia suspensa, 20-35 parts of Taraxacum mongolicum, 5-25 parts of Coptis chinensis, 5-15 parts of Bupleurum chinense, 6-10 parts of Licorice root, 40-45 parts of Star anise, 5-10 parts of Paeonia suffruticosa, 5-12 parts of Radix Trichosanthis, 8-15 parts of Akebia trichosanthis, and 15-20 parts of Pomegranate peel, and sieve the mixture;

[0022] (2) Add 10 times the amount of distilled water, boil at 100°C for 1.5 hours;

[0023] (3) Filter and collect the residue and filtrate 1, add 10 times the amount of distilled water of the residue, boil at 90°C for 1.5 h, filter, and collect filtrate 2;

[0024] (4) combining filtrate 1 and filtrate 2, and concentrating the mixture to a crude drug content of 1 g / mL to obtain a drug concentrate;

[0025] (5) After the concentrated solution is filtered and sterilized, the pH is adjusted to 7, and 0.25-0.5 parts of chicken spleen transfer factor are added and fully dissolved to obtain the drug.

[0026] Preferably, the chicken spleen transfer factor is prepared by the above-mentioned preparation method.

[0027] In a third aspect, the present invention provides a use of a traditional Chinese medicine composition in preparing a medicament for treating chicken Escherichia coli disease, wherein the traditional Chinese medicine composition is composed of the following components in parts by mass:

[0028] Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts;

[0029] The chicken spleen transfer factor is prepared by the above-mentioned method for preparing chicken spleen transfer factor.

[0030] Preferably, the chicken colibacillosis is chicken colibacillosis caused by standard Escherichia coli or mcr-1 positive Escherichia coli;

[0031] The Chinese medicine composition is composed of the following components in parts by mass:

[0032] Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

[0033] In a fourth aspect, the present invention provides a use of a traditional Chinese medicine composition in the preparation of an antibacterial agent for inhibiting mcr-1-positive Escherichia coli, characterized in that the traditional Chinese medicine composition is composed of the following components in parts by mass:

[0034] Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts;

[0035] The chicken spleen transfer factor is prepared by the above-mentioned preparation method.

[0036] Preferably, the Chinese medicine composition consists of the following components in parts by mass:

[0037] Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

[0038] The performance of the Chinese medicine components used in the present invention is explained as follows:

[0039] Gardenia:

[0040] [Source] It is the dried mature fruit of Gardenia jasminoides Ellis, a plant of the Rubiaceae family.

[0041] [Original plant] Gardenia jasminoides Ellis.

[0042] [Harvesting and processing] Harvest the fruits when they are ripe in autumn, remove impurities, and dry them in the sun or at low temperature for later use.

[0043]

Properties

[0044] [Effects] It can relieve heat and relieve restlessness, clear away heat and dampness, cool blood and detoxify.

[0045] Scutellaria baicalensis:

[0046] [Source] It is the dried root of Scutellaria baicalensis Georgi, a plant of the Lamiaceae family.

[0047]

Original plant

[0048] [Harvesting and processing] Dig up the roots in autumn or spring, remove the mud and sand, slice them, and dry them in the sun or at low temperature.

[0049]

Properties

[0050] [Effects] Clears away heat and dampness, purges fire and detoxifies, stops bleeding, and stabilizes the fetus.

[0051] Forsythia:

[0052] [Source] It is the dried fruit of Forsythia suspensa, a plant of the Oleaceae family.

[0053]

Original plant

[0054] Harvesting and Processing: Harvested in autumn when the fruit is just beginning to ripen and still green in color. Remove impurities, steam, and sun-dry. This is commonly known as "Qingqiao." Harvested fully ripe fruit, sun-dried, and impurities removed are commonly known as "Laoqiao."

[0055]

Medicinal properties

[0056]

Effects

[0057] Dandelion:

[0058] [Source] It is the dried whole herb of dandelion, alkali dandelion or various plants of the same genus in the Asteraceae family.

[0059] [Original plant] Dandelion Taraxacum mongolicum Hand.-Mazz. Alkali soil dandelion Taraxacumsinicum Kitag.

[0060]

Harvesting and processing

[0061]

Medicinal properties

[0062] Coptis chinensis:

[0063] [Source] It is the dried rhizome of Coptis chinensis Franch., a plant of the Ranunculaceae family.

[0064]

Original plant

[0065] [Harvesting and processing] Dig up in autumn and winter, remove the soil and fibrous roots, and dry in the sun or at low temperature.

[0066]

Properties

[0067]

Effects

[0068] Bupleurum:

[0069] [Source] It is the dried root of the Umbelliferae plant Bupleurum chinense DC. or Bupleurumscorzonerifolium Willd.

[0070] [Original plant] Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd.

[0071] [Harvesting and processing] Dig up the roots in autumn, remove the mud and fibrous roots, and dry them for later use.

[0072] [Properties] Slightly cold in nature, bitter in taste.

[0073] [Effects] Disperses and reduces fever, relieves liver depression, and raises Yang Qi.

[0074] star anise:

[0075] [Source] It is the dried fruit of Illicium verum Hook.f., a plant of the Magnoliaceae family.

[0076]

Original plant

[0077] [Harvesting and processing] The fruits are picked when they are ripe, and dried in the sun or in an oven for later use.

[0078]

Properties

[0079] [Effects] Warms the middle and regulates Qi, dispels cold and relieves pain, detoxifies and kills insects.

[0080] Trichosanthes:

[0081] [Source] It is the dried root of Trichosanthes kirilowii Maxim., a plant of the Cucurbitaceae family.

[0082]

Original plant

[0083] [Harvesting and processing] Dig up the roots in autumn and winter, remove the skin, and dry them in the sun or at low temperature.

[0084] [Properties] It is slightly cold in nature, sweet and slightly bitter in taste.

[0085]

Effects

[0086] Akebia:

[0087] [Source] It is the dried stem of Akebia quinata (Thunb.) Decne., a plant of the Akebiaceae family.

[0088]

Original plant

[0089] [Harvesting and processing] Harvest the stems in spring and autumn, remove impurities, and dry them in the sun or in the oven for later use.

[0090]

Properties

[0091]

Effects

[0092] Pomegranate peel:

[0093] [Source] It is the dried peel of pomegranate (Punica granatum L.), a plant of the Punicaceae family.

[0094]

Original plant

[0095] [Harvesting and processing] Harvest the fruits when they are ripe in autumn, take the peel, dry it in the sun or at low temperature and set aside.

[0096]

Properties

[0097]

Effects

[0098] Peony bark:

[0099] [Source] It is the dried root bark of Paeonia suffruticosa Andr., a plant of the Ranunculaceae family.

[0100]

Original plant

[0101] [Harvesting and processing] Dig up the roots in autumn, peel off the root bark, and dry them in the sun or at low temperature.

[0102] [Properties] It is slightly cold in nature, bitter and spicy in taste.

[0103]

Effects

[0104] Licorice:

[0105] [Source] It is the dried root and rhizome of the legume plants Glycyrrhiza uralensis, Glycyrrhiza inflata, or Glycyrrhiza glabra. These herbs are commonly known as "Inner Mongolia Licorice," "Xinjiang Licorice," and "European Licorice," respectively.

[0106] [Original plant] Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L.

[0107] [Harvesting and Processing] Dig up in spring and autumn, remove the fibrous roots and stem base, cut into segments of appropriate length, and dry in the sun. Some also remove the outer skin, cut into long segments and dry in the sun, commonly known as "powdered licorice"

[0108]

Medicinal properties

[0109]

Effects

Effects

[0110] The beneficial effects of the present invention are:

[0111] 1. The Chinese medicine composition provided by the present invention has a significant inhibitory effect on both standard Escherichia coli and mcr-1 positive resistant Escherichia coli. Through in vitro antibacterial experiments, the Chinese medicine composition exhibits extremely sensitive and highly sensitive antibacterial effects, especially Chinese medicine composition C, which is significantly superior to the control drug and traditional antibiotic polymyxin B in terms of inhibition zone diameter, effectively solving the problem of difficult treatment of drug-resistant strains.

[0112] 2. Animal experimental results showed that the traditional Chinese medicine composition of the present invention demonstrated extremely high cure rates and overall effectiveness in treating E. coli infection in chickens caused by mcr-1-positive, drug-resistant E. coli. In particular, traditional Chinese medicine composition C achieved a cure rate of 93.3% and an overall effectiveness rate of 100%. Compared with traditional treatments, it significantly reduced the mortality rate in chickens and improved the overall therapeutic effect.

[0113] 3. Through the rational combination of traditional Chinese medicine ingredients, this invention fully utilizes the synergistic effects of the various components, enhancing the antibacterial and immunomodulatory effects. Experimental results show that the omission or substitution of components significantly reduces the efficacy, which verifies the unique formula and irreplaceable nature of the traditional Chinese medicine composition of this invention in the treatment of drug-resistant Escherichia coli.

[0114] Moreover, the Chinese medicine composition of the present invention has the characteristics of multiple components and multiple targets, and can synergistically act through multiple mechanisms such as antibacterial, anti-inflammatory, and immunity enhancement to effectively inhibit the growth of drug-resistant Escherichia coli, avoiding the drug resistance problem caused by the single target action of traditional antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 The encapsulation of the heart and liver of the chickens in the model group 3 days after the infection;

[0116] Figure 2 This is the condition of the heart and liver in experimental group 4 after 5 days of treatment;

[0117] Figure 3 This is the condition of heart and liver encapsulation in the control group and experimental group 3 after 5 days of treatment. DETAILED DESCRIPTION

[0118] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0119] Example 1

[0120] This embodiment provides a Chinese medicine combination drug a for preventing and treating drug-resistant Escherichia coli disease in chickens

[0121] In this medicine, the dosage of Gardenia is 20 parts, the dosage of Scutellaria baicalensis is 20 parts, the dosage of Forsythia suspensa is 30 parts, the dosage of Dandelion is 35 parts, the dosage of Coptis chinensis is 5 parts, the dosage of Bupleurum chinense is 5 parts, the dosage of Licorice root is 6 parts, the dosage of Star Anise is 45 parts, the dosage of Paeonia suffruticosa rubra is 5 parts, the dosage of Radix Trichosanthis is 6 parts, the dosage of Atractylodes lancea is 8 parts, the dosage of Pomegranate peel is 15 parts, and the dosage of Transfer Factor is 0.25 parts.

[0122] The transfer factor is chicken spleen transfer factor, and the preparation method of chicken spleen transfer factor is as follows:

[0123] (a) Raw material pretreatment: Wash the raw materials thoroughly with triple-distilled water, carefully remove impurities such as fascia and fat tissue on the surface, and then rinse repeatedly with sterile saline;

[0124] (2) Ultrasonic wall breaking and homogenization: Cut the cleaned raw materials into small pieces of 1-2 cm, add 2 times the volume of pre-cooled physiological saline (4°C), and grind them into a uniform homogenate using ultrasonic wall breaking technology;

[0125] (3) Repeated freeze-thaw homogenization: The homogenate was quickly frozen in a -20°C refrigerator, then thawed in a 37°C water bath, and the freeze-thaw operation was repeated 6 times. After each complete thawing, the homogenate was immediately homogenized using a tissue homogenizer at high speed to further destroy the cell structure and release the cell contents.

[0126] (4) Low-temperature high-speed centrifugation: The freeze-thawed homogenate was diluted with 3 volumes of pre-cooled physiological saline (4°C), and then centrifuged at 20,000 rpm for 30 minutes at 4°C in a low-temperature centrifuge. The supernatant was collected and pressure-filtered using a 0.45 μm pore size filter membrane to remove larger particles and cell debris.

[0127] (5) Dialysis: The filtrate after filtration was dialyzed using polyethersulfone hollow fiber and the dialyzed solution was continuously circulated for 4 times at 4°C to remove small molecular impurities.

[0128] (6) collecting the filtrate after dialysis and then ultrafiltration using a 5000 molecular weight ultrafiltration membrane;

[0129] (7) The filtrate obtained by ultrafiltration is sterilized by filtration and freeze-dried to obtain chicken spleen transfer factor.

[0130] The drug a is prepared by the following preparation method:

[0131] (1) Grind Gardenia jasminoides, Scutellaria baicalensis, Forsythia suspensa, Taraxacum mongolicum, Coptis chinensis, Bupleurum chinense, Licorice root, Star anise, Paeonia suffruticosa root, Radix Trichosanthis, Akebia trifoliata, and Pomegranate peel, and pass through a No. 2 sieve;

[0132] (2) Add 10 times the amount of distilled water, boil at 100°C for 1.5 hours;

[0133] (3) Filter and collect the residue and filtrate 1, add 10 times the amount of distilled water of the residue, boil at 90°C for 1.5 h, filter, and collect filtrate 2;

[0134] (4) combining filtrate 1 and filtrate 2, and concentrating the mixture to a crude drug content of 1 g / mL to obtain a drug concentrate;

[0135] (5) The concentrated solution was sterilized by filtration using a 0.22 μm filter membrane, and the pH was adjusted to 7. Chicken spleen transfer factor was added and fully dissolved to obtain the Chinese medicine combination drug a.

[0136] Example 2

[0137] This embodiment provides a Chinese medicine combination drug b for preventing and treating drug-resistant Escherichia coli disease in chickens

[0138] In this medicine, the dosage of Gardenia is 10 parts, the dosage of Scutellaria baicalensis is 10 parts, the dosage of Forsythia suspensa is 15 parts, the dosage of Dandelion is 20 parts, the dosage of Coptis chinensis is 25 parts, the dosage of Bupleurum chinense is 15 parts, the dosage of Licorice root is 8 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa rubra is 10 parts, the dosage of Radix Trichosanthis is 12 parts, the dosage of Atractylodes lancea is 15 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0139] The preparation methods of transfer factor and drug b are the same as those in Example 1.

[0140] Example 3

[0141] This embodiment provides a Chinese herbal medicine combination drug c for preventing and treating drug-resistant Escherichia coli disease in chickens

[0142] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Dandelion is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of Transfer Factor is 0.5 parts.

[0143] The preparation methods of transfer factor and drug c are the same as those in Example 1.

[0144] Comparative Example 1

[0145] This comparative example 1 provides a comparative drug a (lacking transfer factor)

[0146] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Dandelion is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, and the dosage of Pomegranate peel is 20 parts.

[0147] The preparation method of the comparative drug is the same as that of Example 1.

[0148] Comparative Example 2

[0149] This comparative example 1 provides a comparative drug b (lacking Gardenia jasminoides and Scutellaria baicalensis)

[0150] In this medicine, the dosage of Forsythia suspensa is 20 parts, the dosage of Dandelion is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes macrocephala is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of Transfer Factor is 0.5 parts.

[0151] The preparation method of the comparative drug is the same as that of Example 1.

[0152] Comparative Example 3

[0153] This comparative example 3 mentions a comparative drug c (lacking Forsythia suspensa and Taraxacum mongolicum)

[0154] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of Transfer Factor is 0.5 parts.

[0155] The preparation method of comparative drug c is the same as that of Example 1.

[0156] Comparative Example 4

[0157] This comparative example 4 provides a comparative drug d (lacking Rhizoma Coptidis and Radix Bupleuri)

[0158] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Dandelion is 30 parts, the dosage of Licorice is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of Transfer Factor is 0.5 parts.

[0159] The preparation method of comparative drug d is the same as that of Example 1.

[0160] Comparative Example 5

[0161] This comparative example 4 provides a kind of comparative medicine e (lack of liquorice and anise)

[0162] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum mongolicum is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0163] The preparation method of comparative drug e is the same as that of Example 1.

[0164] Comparative Example 6

[0165] This comparative example 6 provides a comparative example medicine f (lacking Paeonia suffruticosa and Radix Trichosanthis)

[0166] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Dandelion is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0167] The preparation method of comparative drug f is the same as that of Example 1.

[0168] Comparative Example 7

[0169] This comparative example 7 provides a kind of comparative example medicine g (lacking Akebia and Punica Granatum peel)

[0170] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum mongolicum is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, and the dosage of Transfer Factor is 0.5 parts.

[0171] The preparation method of comparative drug g is the same as that of Example 1.

[0172] Comparative Example 8

[0173] This comparative example 8 provides a comparative drug h (Gardenia jasminoides is replaced by Fructus Aurantii Immaturus and Scutellaria baicalensis is replaced by Atractylodes macrocephala)

[0174] In this medicine, the dosage of Citrus aurantium is 15 parts, the dosage of Atractylodes macrocephala is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum mongolicum is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star anise is 40 parts, the dosage of Paeonia suffruticosa suffruticosa 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0175] The preparation method of comparative drug h is the same as that of Example 1.

[0176] Example 4

[0177] In vitro antibacterial experiments to examine the antibacterial effects of different drugs

[0178] (1) Strain extraction and separation:

[0179] Pathological samples were collected from the market, isolated and PCR identification of drug-resistant genes was performed, and mcr-1 positive drug-resistant Escherichia coli and the standard Escherichia coli strain CMCC.B.44103 were screened out.

[0180] (2) Strain culture:

[0181] The standard strain of E. coli and the positive resistant bacteria were streaked onto LB nutrient agar for activation and cultured at 37°C for 24 hours. A single colony was picked and inoculated into 5 mL of LB liquid medium and cultured in a constant temperature shaker at 180 rpm and 37°C for 24 hours. The bacteria were then counted using the plate count method and diluted with sterile saline to adjust the concentration of the bacterial solution to 1.2 × 10 7 CFU / mL, reserve for future use.

[0182] (3) Experimental testing:

[0183] Absorb 1.2×10 7 Evenly spread 100 μL of the bacterial suspension of the CFU / mL Escherichia coli standard strain and the positive drug-resistant strain on the LB nutrient agar plate. After the surface is slightly dry, place the autoclaved Oxford cup on the plate.

[0184] Drugs were added according to the following experimental groups:

[0185] The control group was added with 200 μL sterile water; the experimental group 1 was added with 5 μg / mL polymyxin B; the experimental group 2 was added with Chinese herbal combination drug a; the experimental group 3 was added with Chinese herbal combination drug b; the experimental group 4 was added with Chinese herbal combination drug c;

[0186] Comparative experimental groups 1-8 were added with the comparative drugs ah prepared in comparative examples 1-8 respectively;

[0187] Three replicate Oxford cups were set up for each bacterial treatment.

[0188] After adding the drug, the culture dish was placed in a 37°C constant temperature incubator and cultured for 24 hours. The diameter of the inhibition zone was measured and expressed as "mean ± standard deviation".

[0189] Table 1 Differences in inhibition zone diameters among different experimental groups

[0190]

[0191]

[0192] The criteria for judging the diameter of the inhibition zone are: diameter ≥ 20mm, extremely sensitive "++++"; 15mm≤diameter<20mm, highly sensitive "+++"; 10mm≤diameter<15mm, moderately sensitive "++"; diameter<10mm is low-sensitivity "+"; no inhibition zone means insensitive "-".

[0193] Analysis of the results in Table 1 shows that polymyxin B in experimental group 1 has a good inhibitory effect on standard Escherichia coli, but a poor inhibitory effect on mcr-1-positive resistant Escherichia coli, which is consistent with expectations.

[0194] Traditional Chinese Medicine Compositions A, B, and C in Experimental Groups 2, 3, and 4 all exhibited excellent inhibitory effects against both standard E. coli and mcr-1-positive, drug-resistant E. coli. Although the inhibitory effect against mcr-1-positive, drug-resistant E. coli declined slightly, it remained at extremely sensitive and highly sensitive levels.

[0195] Among them, Chinese medicine composition c was significantly more effective than Chinese medicine composition a and Chinese medicine composition b. Against standard E. coli, the antibacterial effect of Chinese medicine composition c increased by 45.21% compared to Chinese medicine composition a and by 40.03% compared to Chinese medicine composition b. Against mcr-1-positive, drug-resistant E. coli, the antibacterial effect of Chinese medicine composition c increased by 31.49% compared to Chinese medicine composition a and by 32.04% compared to Chinese medicine composition b. This result demonstrates that the drug prepared according to the formulation of Chinese medicine composition c can more effectively synergize the effects of the various Chinese medicine components and transfer factors, thereby achieving the optimal antibacterial effect.

[0196] The results of comparative experimental group 1 show that transfer factor plays an important role in enhancing the inhibitory effect of the drug of the present invention on E. coli. In particular, for mcr-1 positive drug-resistant E. coli, the loss of transfer factor leads to a significant reduction in the antibacterial effect.

[0197] The results of experimental groups 2 to 7 show that the absence of specific ingredients significantly reduces the antibacterial effect of the control group drug, and its antibacterial effect is significantly weakened compared to the complete Chinese medicine composition C. Especially when targeting mcr-1 positive drug-resistant Escherichia coli, the decline in antibacterial effect is more significant. This result shows that the integrity of the Chinese medicine ingredients of the present invention plays a key role in the effect of the drug in inhibiting Escherichia coli, especially in inhibiting mcr-1 positive drug-resistant Escherichia coli. The absence of certain ingredients may lead to a substantial decrease in drug efficacy. Therefore, ensuring the integrity of Chinese medicine ingredients is an important guarantee for the drug to achieve the best antibacterial effect, especially for drug-resistant strains.

[0198] The results of comparative experimental group 8 showed that when Gardenia jasminoides was replaced with Fructus Aurantii and Scutellaria baicalensis was replaced with Atractylodes macrocephala, the inhibition zone diameter was smaller than that of the experimental group. This result further verified that replacing the components in the traditional Chinese medicine composition of the present invention would not achieve the same efficacy as the composition of the present invention, further demonstrating the irreplaceable and unique nature of the components in the traditional Chinese medicine composition of the present invention.

[0199] Example 5

[0200] Animal experiments to test the therapeutic effects of different drugs on Escherichia coli disease caused by mcr-1 positive Escherichia coli

[0201] (1) The test animals of the present invention were 9-day-old SPF chickens, 250 in total, purchased from Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd.

[0202] (2) The mcr-1 positive Escherichia coli obtained in Example 4 was inoculated into a plate and cultured at 37°C for rejuvenation. The concentration of the bacterial solution was determined by the plate surface live bacteria counting method, so that the concentration of the bacterial stock solution reached 1×10 8 cfu / mL, set aside.

[0203] (3) All chickens were weighed at 10 days of age and randomly divided into a blank control group (30 chickens) and a model group (220 chickens);

[0204] The chickens in the blank control group continued to be raised in a negative pressure isolator, while the chickens in the model group were moved to a temperature and humidity controlled, completely disinfected, relatively closed room to acclimate for 3 days. At 12 days of age, the chickens in the model group were gavaged with 0.5 ml of bacterial stock solution, and the blank group was given the same amount of sterile PBS in the same way. Three days after the infection, 10 chickens were randomly dissected to observe whether the chickens showed typical symptoms such as pericardial perihepatitis (such as Figure 1 As shown in the figure, 3 days after the infection, obvious encapsulation of the heart and liver occurred).

[0205] (4) After successful modeling, the diseased chickens were randomly divided into 7 groups, 30 in each group, namely experimental groups 1-4 and control groups 1-3;

[0206] Experimental groups 1-4 were fed 0.5 ml of water containing polymyxin B, Chinese herbal combination drug a, Chinese herbal combination drug b, and Chinese herbal combination drug c, respectively;

[0207] Control groups 1-3 were fed with water containing control drug a, control drug g, and control drug h, respectively (since the above experiments have shown that the absence of different drugs will lead to a decrease in antibacterial effect, the present invention only selected control drug a, control drug g, and control drug h with better effects among the control drugs for animal experiments);

[0208] The patients were deprived of water at 3:00 pm every day, and the drugs were administered in drinking water at 5:00 pm for 4 hours, and the medication was continued for 5 consecutive days.

[0209] (5) During the experiment, the disease progression of each group of experimental chickens was recorded twice a day at 9:00 am and 4:00 pm. During the experiment, clinical symptoms such as mental state, appetite, and feces were observed and recorded. The cure rate, effective rate, and mortality rate were calculated.

[0210] Criteria for determining efficacy

[0211] Cure: Mental state, diet and defecation return to normal, and symptoms completely disappear;

[0212] Effective: Mental state, diet, and defecation improved, and symptoms were significantly alleviated;

[0213] Ineffective: No improvement in symptoms or even death; autopsy reveals varying degrees of pericarditis, perihepatitis and other lesions.

[0214] Table 2 The therapeutic effects of different experimental groups on mcr-positive drug-resistant Escherichia coli disease

[0215]

[0216]

[0217] First, if Figure 2 This is the result after treatment of experimental group 4. Combined with actual observations, it can be seen that after treatment, the symptoms of pericarditis and perihepatitis disappeared and returned to normal. At the same time, the mental state and stool were normal.

[0218] Figure 3 This is the result after treatment of control experimental group 3. It can be seen that mild pericarditis and perihepatitis symptoms still exist after treatment.

[0219] From the results in Table 2, we can see that

[0220] Experimental Group 1: The cure rate of this group was only 13.3%, the effective rate was 16.7%, and the overall effective rate was only 30%. This shows that polymyxin B is not very effective in treating chickens infected with mcr-1-positive drug-resistant E. coli.

[0221] Experimental Groups 2, 3, and 4 (TCM compositions a, b, and c): The therapeutic effects of these groups were significantly better than those of Experimental Group 1, especially Experimental Group 4 (TCM composition c). The cure rates of Experimental Groups 2 and 3 were 73.3% and 76.7%, respectively, with effective rates of 1.00% and 1.33%, respectively, and total effective rates of 83.3% and 90%, respectively. The cure rate of Experimental Group 4 was as high as 93.3%, with an effective rate of 0.67% and a total effective rate of 100%, demonstrating significant therapeutic effects. TCM composition c demonstrated the best efficacy in treating mcr-1-positive E. coli infections, which is consistent with its strong antibacterial effect shown in Table 1.

[0222] Control groups 1-3: These groups used different control drugs a, g, and h, respectively. Their cure rates and overall effectiveness rates were generally lower than those of the experimental groups. Control group 1 had a cure rate of 26.7% and an overall effectiveness rate of 56.7%; control group 2 had a cure rate of 23.3% and an overall effectiveness rate of 53.3%; and control group 3 had a cure rate of 30% and an overall effectiveness rate of 63.3%. These results further demonstrate the uniqueness and efficacy of the traditional Chinese medicine composition of this invention. The integrity and synergistic effects of the ingredients are crucial in enhancing therapeutic efficacy; either the omission or substitution of some ingredients will significantly reduce efficacy.

[0223] In summary, this study showed that Chinese medicine composition C has a significant therapeutic effect in the treatment of mcr-1 positive Escherichia coli infection, and its efficacy is significantly better than polymyxin B and other comparison drugs.

Claims

1. A Chinese medicinal composition for preventing and treating chicken Escherichia coli disease, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts; The preparation method of the chicken spleen transfer factor comprises the following steps: (1) Wash the raw materials thoroughly with triple-distilled water, carefully remove surface impurities, and then rinse repeatedly with sterile saline; (2) Cut the cleaned raw materials into small pieces of 1-2 cm, add 2 times the volume of 4°C pre-cooled physiological saline, and grind them into a uniform homogenate using ultrasonic wall breaking technology; (3) The homogenate was quickly frozen in a -20°C refrigerator, then thawed in a 37°C water bath. The freeze-thaw operation was repeated 6 times. After each complete thawing, the homogenate was immediately homogenized and crushed using a tissue homogenizer at high speed to further destroy the cell structure and release the cell contents. (4) The freeze-thawed homogenate was diluted with 3 volumes of 4°C pre-cooled physiological saline, and then centrifuged at 20,000 rpm for 30 minutes at 4°C using a low-temperature centrifuge. The supernatant was collected and pressure filtered using a 0.45 μm pore size filter membrane to remove larger particles and cell debris. (5) The filtrate was dialyzed using a polyethersulfone hollow fiber at 4°C for 4 consecutive cycles to remove small molecular impurities; (6) Collect the filtrate after dialysis and then ultrafilter it using a 5000 molecular weight ultrafiltration membrane; (7) The filtrate obtained by ultrafiltration is filtered and sterilized, and then freeze-dried to obtain chicken spleen transfer factor; The chicken colibacillosis is caused by standard Escherichia coli or mcr-1 positive Escherichia coli.

2. The Chinese medicine composition according to claim 1, characterized in that The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Chicken Spleen Transfer Factor: 0.5 parts.

3. A method for preparing a drug for preventing and treating chicken Escherichia coli disease, characterized in that: The preparation method comprises the following steps: (1) Grind 10-20 parts of Gardenia jasminoides, 10-20 parts of Scutellaria baicalensis, 15-30 parts of Forsythia suspensa, 20-35 parts of Taraxacum mongolicum, 5-25 parts of Coptis chinensis, 5-15 parts of Bupleurum chinense, 6-10 parts of Licorice root, 40-45 parts of Star anise, 5-10 parts of Paeonia suffruticosa, 5-12 parts of Radix Trichosanthis, 8-15 parts of Akebia trifoliata, and 15-20 parts of Pomegranate peel, and sieve; (2) Add 10 times the amount of distilled water, boil at 100°C for 1.5 hours; (3) Filter and collect the residue and filtrate 1, add 10 times the amount of distilled water of the residue, boil at 90°C for 1.5 hours, filter, and collect filtrate 2; (4) Filtrate 1 and filtrate 2 were combined and concentrated to a crude drug content of 1 g / mL to obtain a drug concentrate; (5) After filtering and sterilizing the concentrated solution, the pH is adjusted to 7, 0.25-0.5 parts of chicken spleen transfer factor is added, and after fully dissolving, the drug is obtained; The chicken spleen transfer factor is prepared by the preparation method described in claim 1.

4. Use of a Chinese medicine composition in preparing a medicament for treating chicken Escherichia coli disease, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts; The chicken spleen transfer factor is prepared by the preparation method described in claim 1.

5. The use according to claim 4, characterized in that The chicken colibacillosis is caused by standard Escherichia coli or mcr-1 positive Escherichia coli; The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Chicken Spleen Transfer Factor: 0.5 parts.

6. Use of a traditional Chinese medicine composition in the preparation of an antibacterial agent for inhibiting mcr-1 positive Escherichia coli, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts; The chicken spleen transfer factor is prepared by the preparation method described in claim 1.

7. The use according to claim 6, characterized in that The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Chicken Spleen Transfer Factor: 0.5 parts.

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